Clinical Studies Of Abnormal Host Defense

宿主防御异常的临床研究

• 批准号: 7732421
• 负责人: JOHN I GALLIN
• 金额: $ 16.95万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732421
• 关键词: Aftercare; Age; Alexa fluor 546; Amphotericin B; Antibiotics; Antifungal Agents; Aspergillus; Aspergillus fumigatus; B-Lymphocytes; Biological; Biomedical Research; Bulla; Candida albicans; Carbohydrates; Cell Line; Cell Nucleus; Cell physiology; Cells; Chelating Agents; Chemotaxis; Child; Chronic Granulomatous Disease; Clinical; Clinical Research; Code; Collaborations; Complement; Complex; Confocal Microscopy; Cryptococcus neoformans; Cyclic Neutropenia; Cytoplasmic Granules; DAPI; DNA Sequence; Data; Defect; Development; Event; Exhibits; Exons; Extramural Activities; F-Actin; Fluconazole; Frequencies; Functional disorder; Future; Genes; Genetic; Genotype; Germination; Goals; Growth; Growth Factor; Host Defense; Host Defense Mechanism; Human; Human Herpesvirus 4; Hydrogen Peroxide; Hyphae; IRAK4 gene; Immune System Diseases; Immune system; Immunocompromised Host; In Vitro; Infection; Infectious Agent; Interferon Type II; Interferons; Intramural Research Training Award; Iron; Iron Chelating Agents; Itraconazole; Job' s Syndrome; Laboratories; Lactoferrin; Left; Leukocyte Adhesion Deficiency; Leukocyte-Adhesion Deficiency Syndrome; Life; Limulus; Lipid A; Lobe; Localized; Longitudinal Studies; Lower Respiratory Tract Infection; Membrane; Metabolic Pathway; Metals; Microbe; Microscopic; Missense Mutation; Modeling; Molecular; Monitor; Morphology; Mutation; Mycoses; NADPH Oxidase; Neutropenia; Nonsense Mutation; Nuclear; Oral; Organism; Pathogenesis; Patients; Phagocytes; Pharmaceutical Preparations; Play; Prevention; Production; Property; Prophylactic treatment; Publishing; Purpose; Recording of previous events; Recruitment Activity; Recurrence; Regulation; Research; Resistance; Resources; Role; Shapes; Sister; Skin; Staining method; Stains; Structure; Subgroup; Surface; Syndrome; System; Testing; Therapeutic; Today; Twin Multiple Birth; USA Georgia; Ulcer; United States National Institutes of Health; Universities; Virulence; Western Asia Georgia; antimicrobial peptide; base; chediak-higashi syndrome; cohort; cytokine; fungus; gallin; human IRAK4 protein; in vivo; killings; medical schools; mortality; neutrophil; neutrophil cytosol factor 40K; neutrophil cytosol factor 67K; novel; novel therapeutics; oral infection; pathogen; receptor internalization; response; septic; uptake

The LHD has a long history of investigating patients with abnormalities of phagocytic cell function. These studies include the delineation of clinical, functional, and molecular defects of patients with neutrophil specific granule deficiency, chronic granulomatous disease (CGD), leukocyte adhesion deficiency (LAD), the syndrome of hyperimmunoglobulinE and recurrent infections (Jobs Syndrome) and IRAK4 deficiency. Large cohorts of these patients have been recruited over the years and represent a unique national resource for biomedical research at the NIH. Currently we follow over 150 patients with CGD, about 40 patients with Jobs syndrome, and 30 patients with other phagocyte dysfunction syndromes, including LAD, cyclic neutropenia, neutrophil specific granule deficiency, Chediak-Higashi syndrome, IRAK4 deficiency and NEMO deficiency. We now have EB virus transformed B cells from most of our patients and we have been pleased to share these B cell lines with other intramural or extramural colleagues. We continue to monitor and expand these cohorts of patients who serve as models for long term studies of the clinical consequences of the immune dysfunction in humans. In 2008 we continued our studies of the importance of lactoferrin in protecting against Aspergillus fumigatus infection, the most common infectious cause of mortality in CGD patients today. We found that while CGD PMN are unable to kill Aspergillus hyphae, their ability to arrest the growth of conidia was identical to that of normal PMN showing a role for nonoxidative mechanisms in host defenses against this organism. We then showed that the neutrophil secretory product, lactoferrin, inhibits conidial germination by sequestering iron, a critical growth factor. A postbac IRTA, Anna R. Cruz (who recently left the lab to attend medical school), has continued studying the growth inhibitory properties of iron chelating drugs against Aspergillus fumigatus, alone and in combination with first line antibiotics such as amphotericin B, itraconazole, and fluconazole and demonstrated antifungal synergy in some combinations. Further studies have demonstrated significant antifungal activity of some iron chelators against other fungi (Cryptococcus neoformans, Candida albicans) suggesting an important new avenue for prevention and treatment of fungal infections common to immunocompromised patients. (Anna R. Cruz, 50% effort, Kol Zarember, 30% effort). In 2008 we published other studies (in collaboration with Janyce Sugui and June Kwong Chung, NIH/NIAID/LCID) investigating responses of Aspegillus to attack by normal and CGD neutrophils. We found differential regulation of many genes putatively involved in metal uptake and a variety of other fungal metabolic pathways that may be promising targets for new therapeutic approaches. Putative virulence genes identified by this approach have been deleted and we are pursuing whether they play a role in infection or resistance to human PMN attack. DNA sequencing of CGD patients continues to reveal new information about molecular components of the NADPH oxidase system. In 2008 we completed the sequencing of p47phox and gp91phox genes from about 200 living and deceased CGD patients. Nearly all patients with p47phox deficiency exhibited the same GT deletion in exon 1. Interestingly, analysis of gp91phox deficiency revealed 77 different mutations or deletions randomly distributed through out the coding regions with 32 novel mutations detected. Mutations in p67phox and p40phox CGD patients are currently being analyzed. The sequencing of gp91phox in these patients has revealed an important correlation between genotype and potential efficacy of one of the main prophylactic treatments available in the management of CGD. Previously (N. Engl. J. Med. 324:509-516, 1991), we had shown that treatment of CGD patients with IFN-γ reduced the overall frequency of infections and certain CGD subgroups (p47phoxCGD and a subset of the patients with gp91phox CGD ) responded to IFN γ both in vitro and in vivo whereas others did not (PNAS, 85:4874-4878, 1988). With the sequence data now available, a retrospective analysis revealed that the gp91phox subgroup responsive to IFN-γ represented patients with missense mutations in gp91phox while the gp91phox subgroup unresponsive to IFN-γ represented patients with nonsense mutations. We have recently repeated those studies in vitro, a study that included over 650 analyses of H2O2 production, and showed that CGD patients with missense mutation in gp91phox respond to IFN-γ in vitro (p = 0.045) while patients with nonsense mutations in gp91phox fail to respond to IFN-γ (p = 0.419). Moreover, CGD patients with a GT deletion in exon 1 of p47phox also respond to IFN-γ (p = 0.0009). These findings provide a genetics-based rationale for predicting the efficacy of IFN-γ therapy in the treatment of patients with CGD and may have important therapeutic implications for guiding future therapy. (Gallin, Malech, Holland, Kuhns). Over the past year, the laboratory has expanded its study of Granulibacter bethesdensis, a recently described bacterial pathogen of CGD patients. Granulibacter is remarkably hypostimulatory of the innate immune system, both in terms of weak activation of the NADPH oxidase and poor stimulation of cytokine secretion. With collaborators at the University of Georgia Complex Carbohydrate Research Center, we are purifying and characterizing the biological activity and structure of the LPS. We have isolated a fraction that appears to represent the Lipid A of this organism and that has strongly stimulatory activity in the limulus LPS test but fails to activate human PMN in-keeping with our findings that this microbe may avoid host defenses by using molecular stealth. We have also found that G. bethesdensis is remarkably resistant to complement and antimicrobial peptides. Further studies are underway to understand the pathogenesis of this organism in CGD patients and host defenses against it (Zarember, 40% effort, Anna R. Cruz 50% effort and contributions by David E. Greenberg, LCID). In 2008 we studied two Qatari sisters (5 & 12 yrs.) with an undescribed neutrophil dysfunction. The sisters had frequent severe skin and mucosal ulcerations, recurrent upper and lower respiratory tract infections, and neutropenia. The younger sister had repeated oral infections that ultimately resulted in development of oral strictures. A twin sister of the younger child who had a similar presentation died at age 3 years from a septic event. Although degranulation and staphylocidal activity were not dramatically altered, a profound chemotactic defect in vitro was detected. Microscopic examination of neutrophils from both sisters showed abnormal morphology with herniation of nuclear lobes into membrane-enclosed surface blebs. Neutrophils from both sisters failed to undergo a shape change after treatment with fMLF. Moreover, herniation of nuclear lobes coincided with retraction of the granules into the central region of the cells, resulting in an agranular region in the periphery of the cell. Confocal microscopy, using Alexa Fluor 546-phalloidin to identify filamentous actin and DAPI to localize nuclei, showed that cells with herniated nuclear lobes exhibited increased levels of filamentous actin compared to either patient cells with normal nuclear lobes or to normal neutrophils. Using FACS, a 4-5-fold increase in F-actin staining was detected in patient cells compared to normal controls. The abnormal nuclear morphology, profound defects in chemotaxis and in receptor internalization, and cytoskeletal anomalies detected in these two sisters with recurrent infections represents a novel defect in neutrophils that is likely an autosomal recessive mutation. (Kuhns, Uzel, Gallin and Holland).

LHD在研究吞噬细胞功能异常的患者方面有悠久的历史。这些研究包括嗜中性粒细胞特异性颗粒缺乏症患者的临床，功能和分子缺损的描述，慢性颗粒疾病（CGD），白细胞粘附缺乏（LAD），高免疫球蛋白和复发性感染（工作综合症）的综合征，综合症综合症（工作综合症）和伊拉克综合症。这些年来，这些患者的大量人群已被招募，代表了NIH的独特国家生物医学研究资源。目前，我们关注150多名CGD患者，约40例工作综合征患者以及30例其他吞噬细胞功能障碍综合症患者，包括LAD，环状中性粒细胞减少症，中性粒细胞特异性颗粒缺乏症，Chediak-Higashi综合征，IRAK4缺乏症和Nemo缺乏症。现在，我们拥有EB病毒从大多数患者中转化的B细胞，我们很高兴与其他壁内或壁外同事共享这些B细胞系。我们继续监视和扩展这些同类的患者，这些患者是长期研究人类免疫功能障碍的临床后果的模型。 2008年，我们继续研究乳铁蛋白在预防曲霉感染中的重要性，这是当今CGD患者死亡率最常见的感染原因。我们发现，尽管CGD PMN无法杀死曲霉的菌丝，但它们阻止分生孢子的生长与正常PMN的生长能力相同，在宿主防御剂中对这种生物体的防御措施表现出了非氧化机制的作用。然后，我们表明嗜中性粒细胞分泌产物乳铁蛋白通过隔离铁（一种关键生长因子）抑制分生孢子发芽。 A postbac IRTA, Anna R. Cruz (who recently left the lab to attend medical school), has continued studying the growth inhibitory properties of iron chelating drugs against Aspergillus fumigatus, alone and in combination with first line antibiotics such as amphotericin B, itraconazole, and fluconazole and demonstrated antifungal synergy in some combinations.进一步的研究表明，某些铁螯合剂对其他真菌（隐孢子虫，白色念珠菌）的显着抗真菌活性，这表明预防和治疗免疫功能低下患者常见的真菌感染的重要新途径。 （安娜·R·克鲁兹（Anna R. Cruz），努力为50％，科尔·扎伦贝（Kol Zarember），努力30％）。 2008年，我们发表了其他研究（与Janyce Sugui和June Kwong Chung合作，NIH/NIAID/LCID）研究了Aspegillus对正常和CGD中性粒细胞攻击的反应。 我们发现，对金属摄取的许多基因和许多其他真菌代谢途径的差异调节，这可能是新治疗方法的有希望的靶标。通过这种方法鉴定出的推定毒力基因已被删除，我们正在追求它们是在感染还是对人类PMN攻击的抵抗中发挥作用。 CGD患者的DNA测序继续揭示有关NADPH氧化酶系统分子成分的新信息。 在2008年，我们完成了来自约200名活着和已故CGD患者的P47Phox和GP91Phox基因的测序。几乎所有患有P47PHOX缺乏症的患者在外显子1中均表现出相同的GT缺失。有趣的是，对GP91Phox缺乏症的分析揭示了77种不同的突变或缺失，随机通过检测到32个新型突变的编码区域随机分布。目前正在分析p67phox和p40phox CGD患者的突变。 这些患者中GP91Phox的测序表明，基因型与CGD管理中主要预防性治疗之一的潜在疗效之间存在重要的相关性。 以前（N.Engl。J.Med。324：509-516，1991），我们表明，IFN-γ的CGD患者的治疗降低了感染和某些CGD亚组的总体频率和某些CGD亚组（p47phoxCGD（p47phoxCGD）（p47phoxCGD和患者的子集）对GP91Phox CGD患者的子集对INγ和IngO均响应（PN）其他VIV（PN） 85：4874-4878，1988）。通过现在可用的序列数据，回顾性分析表明，对IFN-γ的GP91Phox亚组反应反应IFN-γ，代表了GP91Phox中具有错义突变的患者，而GP91Phox亚组对IFN-γ的反应无反应代表了无义突变的患者。 我们最近在体外重复了这些研究，该研究包括超过650种H2O2生产分析，并表明GP91Phox中具有错义突变的CGD患者在体外对IFN-γ作出反应（P = 0.045），而GP91PHOX中无义突变的患者无法应对IFN-γ（P = 0.419）。 此外，p47phox外显子1中具有GT缺失的CGD患者也对IFN-γ做出反应（p = 0.0009）。 这些发现提供了一种基于遗传学的基本原理，以预测IFN-γ治疗在治疗CGD患者中的功效，并且可能对指导未来的治疗具有重要的治疗意义。 （Gallin，Malech，Holland，Kuhns）。 在过去的一年中，该实验室扩大了对肉芽杆菌贝塞斯丁斯（Bethesdensis）的研究，这是最近描述的CGD患者的细菌病原体。颗粒杆菌对先天性免疫系统的极不受欢迎，这是因为NADPH氧化酶的激活和细胞因子分泌的刺激不良。 与佐治亚大学复杂碳水化合物研究中心的合作者一起，我们正在净化和表征LP的生物学活性和结构。我们分离了一个似乎代表该生物体的脂质A的部分，并且在Limulus LPS测试中具有强烈的刺激活性，但未能通过我们的发现激活人类PMN的PMN，即该微生物可以通过使用分子隐身来避免宿主防御。 我们还发现，贝塞斯丁氏菌对补体和抗菌肽具有明显抗性。正在进行进一步的研究，以了解该生物在CGD患者中的发病机理和宿主防御措施（Zarember，40％的努力，Anna R. Cruz 50％的努力和David E. Greenberg，LCID的贡献）。 在2008年，我们研究了两个卡塔里姐妹（5岁和12年），并具有未描述的中性粒细胞功能障碍。姐妹经常出现严重的皮肤和粘膜溃疡，复发性上下呼吸道感染以及中性粒细胞减少症。 妹妹重复口腔感染，最终导致口腔狭窄的发展。 一个年幼的孩子的双胞胎姐姐，他的演讲类似，享年3岁。 尽管脱粒和葡萄球菌活性并未显着改变，但在体外检测到了深刻的趋化缺陷。 对两个姐妹的中性粒细胞的微观检查均显示出异常的形态，核叶催眠到膜粘附的表面爆炸中。两位姐妹的中性粒细胞在用FMLF治疗后未能发生变化。 此外，核叶的疝与颗粒缩回细胞中心区域相吻合，从而导致细胞周围的植物区域。 共聚焦显微镜使用Alexa Fluor 546-甲状腺素鉴定丝状肌动蛋白和DAPI来定位核，表明与正常核叶或正常性中性粒细胞的患者细胞相比，具有椎间盘核叶的细胞表现出丝状肌动蛋白的水平增加。 使用FACS，与正常对照组相比，患者细胞中检测到F-肌动蛋白染色的4-5倍。异常的核形态，趋化性和受体内在化的深刻缺陷以及在这两个姐妹中检测到的具有复发性感染的姐妹的细胞骨架异常代表中性粒细胞中的新缺陷，可能是一种常染色体隐性突变。 （Kuhns，Uzel，Gallin和Holland）。

项目成果

Mutational analysis of patients with p47-phox-deficient chronic granulomatous disease: The significance of recombination events between the p47-phox gene (NCF1) and its highly homologous pseudogenes.

p47-phox 缺陷型慢性肉芽肿病患者的突变分析：p47-phox 基因 (NCF1) 与其高度同源的假基因之间重组事件的意义。

• DOI: 10.1016/s0301-472x(00)00646-9
• 发表时间: 2001
• 期刊: Experimental hematology
• 影响因子: 2.6
• 作者: Vazquez,N;Lehrnbecher,T;Chen,R;Christensen,BL;Gallin,JI;Malech,H;Holland,S;Zhu,S;Chanock,SJ
• 通讯作者: Chanock,SJ

Short stature in partially corrected X-linked severe combined immunodeficiency--suboptimal response to growth hormone.

• DOI: 10.1515/jpem.2008.21.11.1057
• 发表时间: 2008-11
• 期刊: Journal of pediatric endocrinology & metabolism : JPEM
• 作者: De Ravin SS;Shum E;Zarember KA;Rezvani G;Rosenfeld RG;Stratakis CA;Malech HL
• 通讯作者: Malech HL

Taming clinical research's paper tigers.

驯服临床研究的纸老虎。

• DOI: 10.1158/1078-0432.ccr-06-1095
• 发表时间: 2006
• 期刊: Clinical cancer research : an official journal of the American Association for Cancer Research
• 作者: Gallin,JohnI

Response to: "Rescuing the NIH before it is too late".

回应：“在为时已晚之前拯救 NIH”。

• DOI: 10.1172/jci28894
• 发表时间: 2006
• 期刊: The Journal of clinical investigation
• 作者: Alexander,DuaneF;Alving,BarbaraM;Battey,JamesF;Berg,JeremyM;Collins,FrancisS;Fauci,AnthonyS;Gallin,JohnI;Grady,PatriciaA;Hodes,RichardJ;Hrynkow,SharonH;Insel,ThomasR;Jones,JackF;Katz,StephenI;Landis,StoryC;Li,Tin
• 通讯作者: Li,Tin